ABSTRACTEthylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.

f1: Changes in rubber latex parameters after different treatments.The fresh yield (a), dry matter (b), water content (c) and flow time (d) for latex collected from rubber trees treated with ethylene (3% ethephon, marked with black rectangle) and ddH2O (white) for 0, 3 and 5 days are presented. Total rubber particles from untreated plants (e, day 0) and plants treated with ddH2O (f) and ethylene (g) for 3 days were examined under SEM.

Mentions:
The effects of ethylene stimulation are more obvious in virgin plants than in mature trees91213. A low-frequency tapping procedure referred to as “half-spiral and once tapping in three days plus ethrel” is the recent method of choice for latex collection14. Therefore, we used this procedure on virgin plants in this study. Overall, ethylene stimulation sharply improved the yield of fresh latex (Fig. 1a) and dry matter (Fig. 1b). Before treatment, approximately 5 ml of latex was recovered per plant. After ethylene treatment for 3 and 5 days, the volume of recovered latex reached ∼17 and 12 ml, respectively. Compared with the control, ddH2O-treated plants also exhibited a significant increase in latex (Fig. 1a), which were attributed to the mechanical effects of wounding caused by tapping15. Consistent with reported results7916, ethylene also improved the latex water content (Fig. 1c) and markedly prolonged the latex flow time (Fig. 1d). Hevea rubber particles are spherical or pear-shaped, measuring approximately 0.02–3.0 μm diameter and, based on their bimodal size distribution, including both large and small rubber particles (LRPs and SRPs, respectively)1718. In this study, the accumulation of SRPs was visibly induced by ethylene stimulation (Fig. 1e–g). In control, LRPs were the most frequently observed rubber particles (Fig. 1e). After tapping, the number of SRPs increased substantially (Fig. 1f). Notably, almost all examined rubber particles were SRPs following three days of ethylene (E-3) treatment (Fig. 1g). Recently, SRPP protein in SRPs has been reported to play a more important role than REF does in LRP natural rubber biosynthesis51819. Taken with these reports51819, our observations suggest that the ethylene stimulation of rubber latex can likely be attributed to the generation of SRPs.

f1: Changes in rubber latex parameters after different treatments.The fresh yield (a), dry matter (b), water content (c) and flow time (d) for latex collected from rubber trees treated with ethylene (3% ethephon, marked with black rectangle) and ddH2O (white) for 0, 3 and 5 days are presented. Total rubber particles from untreated plants (e, day 0) and plants treated with ddH2O (f) and ethylene (g) for 3 days were examined under SEM.

Mentions:
The effects of ethylene stimulation are more obvious in virgin plants than in mature trees91213. A low-frequency tapping procedure referred to as “half-spiral and once tapping in three days plus ethrel” is the recent method of choice for latex collection14. Therefore, we used this procedure on virgin plants in this study. Overall, ethylene stimulation sharply improved the yield of fresh latex (Fig. 1a) and dry matter (Fig. 1b). Before treatment, approximately 5 ml of latex was recovered per plant. After ethylene treatment for 3 and 5 days, the volume of recovered latex reached ∼17 and 12 ml, respectively. Compared with the control, ddH2O-treated plants also exhibited a significant increase in latex (Fig. 1a), which were attributed to the mechanical effects of wounding caused by tapping15. Consistent with reported results7916, ethylene also improved the latex water content (Fig. 1c) and markedly prolonged the latex flow time (Fig. 1d). Hevea rubber particles are spherical or pear-shaped, measuring approximately 0.02–3.0 μm diameter and, based on their bimodal size distribution, including both large and small rubber particles (LRPs and SRPs, respectively)1718. In this study, the accumulation of SRPs was visibly induced by ethylene stimulation (Fig. 1e–g). In control, LRPs were the most frequently observed rubber particles (Fig. 1e). After tapping, the number of SRPs increased substantially (Fig. 1f). Notably, almost all examined rubber particles were SRPs following three days of ethylene (E-3) treatment (Fig. 1g). Recently, SRPP protein in SRPs has been reported to play a more important role than REF does in LRP natural rubber biosynthesis51819. Taken with these reports51819, our observations suggest that the ethylene stimulation of rubber latex can likely be attributed to the generation of SRPs.

ABSTRACTEthylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.